**3. Use of** *Lachancea thermotolerans* **and** *Hanseniaspora* **spp. at industrial scale**

The use of a new non-*Saccharomyces* strain requires a lot of experimental research in the laboratory, but also several years of pilot, semi-industrial and industrial-scale trials. **Table 1** details the fermentations, years, wineries, regions, varieties, volumes, controls, and pH effects of selected *Lachancea thermotolerans* strains L31 and A54, currently under industrial evaluation by Lallemand. The strains were tested on white and red grape varieties to see the implantation and performance of acidification on settled white must, but also on crushed red grapes with skins and seeds. Volumes ranged from 500 to 12,000 in white musts and from 1,000 kg to 15,000 kg in crushed red grapes.

In all conditions, acidification was quite effective, even in crushed grapes where the high presence of indigenous yeasts can affect the implantation by reducing the prevalence of the Lt strain. It is interesting to highlight that acidification is effective in varieties with low pHs such as Albariño (3.1) and varieties with high initial pH


#### **Table 1.**

*Performance of* Lachancea thermotolerans *L31 & A54 strains on several semi-industrial trials.*

*pH Control and Aroma Improvement Using the Non-*Saccharomyces Lachancea*… DOI: http://dx.doi.org/10.5772/intechopen.100538*

such as Airén or Tempranillo (3.75–4.20). In terms of potential alcohol, the varieties showed alcoholic strengths ranging from 11 to 12% vol. in the whites and 14–15% in the reds.

Volatile acidity was quite moderate and ranged from 0.38 to 0.46 g/L. The other fermentative volatiles were at normal values for the wines, only the ethyl lactate content was higher than the Sc controls (40–50 mg/L) due to intense lactic acid production, but below the sensory threshold for this ester (150 mg/L) [22].

It is important to note that when Lt strains are used on an industrial scale on real musts or crushed grapes it is important to keep the total SO2 concentration below 20 mg/L. Otherwise, Lt implantation and development can be seriously affected. The typical acidification pattern shows maximum lactic acid production at the beginning of fermentation (days 3–6, **Figure 5**) depending on inoculation rate, temperature, nutrients, and must composition [22, 23, 45].

It can be observed how the high pH typical of varieties such as Tempranillo in warm areas is deleterious to wine quality, not only producing chemical and microbial instability but also making sulfites inefficient due to low molecular SO2 levels. The natural biological acidification of Lt produces pH reductions from 4.0 to 3.5 or less resulting in molecular SO2 levels increasing from <0.4 (dangerous) to >0.8 (safe) [25]. It should also be noted that lactic acid is a stable acid that cannot be altered or metabolized by microorganisms during wine aging. In addition, at high doses (>4 g/L) it inhibits malolactic fermentation, which can be interesting to maintain extra acidity and protect the freshness in wines from warm areas [46].

From a sensory point of view, biological acidification produces a citric freshness, which can be very crispy at high concentrations but can never be perceived as dairy acidity. This is because the milky profile of malolactic fermentation and fermented milk comes from some secondary metabolites such as acetoin or diacetyl that are found in low concentrations in Lt fermentations.

The typical sensory profile of Lt normally shows increased freshness with improved acidity (**Figure 6**) which, depending on the level of acidification, can be somewhat unbalanced and crispy. This can be controlled by the timing of Sc inoculation in sequential fermentation or, subsequently, by blending Lt wines with Sc wines. Even when Lt does not have a strong impact on the aroma, the profile is fresh, fruity, and pleasant. The body in the wines is similar to that of Sc, but, as noted above, specific strains have effects on palatability.

#### **Figure 5.**

*Typical pH evolution in industrial fermentations driven by* Lachancea thermotolerans*. The gradient color scale shows the safety of wines in terms of microbial and chemical stability as a function of pH.*

#### **Figure 6.**

Additionally, we have compared in Airen fermentations the effect of 72 h of biological acidification with Lt (2 strains: L31 and Laktia from Lallemand) with chemical acidification using 1.5 g/L tartaric acid. Natural biological acidification produced the same effect on pH without using chemical additives [47]. Furthermore, chemical stability is higher due to the high potassium salts precipitation produced during chemical acidification with tartaric acid.

Concerning the use of *Hanseniaspora* spp. on an industrial scale, the most important species are *Hanseniaspora vineae* and *H. opuntiae*, although *H. uvarum* has also been used to some extent. We have experience fermenting Albillo (*Vitis vinifera* L.) white variety with *H. uvarum* in stainless steel and oak barrels to produce white wines aged on lees or blends of Albillo and Tempranillo (*Vitis vinifera* L.) to produce rosé wines (**Table 2**). Moreover, we have fermented must from Airen (*Vitis vinifera* L.),


#### **Table 2.**

*Performance of* Hanseniaspora *spp. on several semi-industrial trials.* Hanseniaspora vineae *(Hv),*  Hanseniaspora opuntiae *(Ho).*

*pH Control and Aroma Improvement Using the Non-*Saccharomyces Lachancea*… DOI: http://dx.doi.org/10.5772/intechopen.100538*

#### **Figure 7.**

*Comparative sensory spider net of fermentations with* Hanseniaspora vineae/opuntiae *and* Saccharomyces cerevisiae*.*

a neutral flat grape variety, in large stainless-steel tanks using *H. opuntiae*. This species enabled the production of wines with more body, better palatability, and floral aroma.

The formation of terpenes and floral esters by *Hanseniaspora* spp. has an interesting impact on the sensory profile, especially with neutral grape varieties such as Airén or Albillo that express fruitier and more floral wines with greater aromatic freshness. In addition, a positive effect on color can be found in rosé wines with higher anthocyanin contents in fermentations with Hv and especially some acylated derivatives [48]. **Figure 7** shows the typical sensory profile of *Hanseniaspora* spp. compared to *Saccharomyces cerevisiae*.

#### **4. Biocompatibility**

Lt and Hv/Ho can be used in mixed fermentations or independent fermentations, subsequently blending both wines in appropriate quantities. When used in mixed fermentations, biocompatibility must be taken into account due to the special sensitivity of *Hanseniaspora* to vitamins such as thiamine and pantothenate or nitrogen contents. Nutritional deficits can lead to the low formation of acetate esters and terpenes with the consequence of a low impact on the aroma. A similar situation is observed in *Lachancea thermotolerans* in which nutritional imbalances affect implantation and development of the yeast population and therefore low acidification compromising the effect on pH. Lower acidification has been observed in ternary fermentations with Lt and Hv sequentially followed by Sc under standard nutritional conditions [45]. The development of further research to carefully optimize the nutritional and physicochemical conditions (temperature, SO2, pH) for interspecies compatibility will be a key parameter for the successful application of this biotechnology.

#### **5. Conclusion**

The combined use of *Hanseniaspora* spp. (*vineae* or *opuntiae*) with *Lachancea thermotolerans* in mixed fermentations subsequently finished sequentially by

*Saccharomyces* or the independent use of them and later blending their wines is interesting biotechnology to improve flat neutral varieties by increasing acidity, aroma, body, and color, and thus improving the sensory profile and freshness. Several considerations have been described to achieve successful fermentations in terms of nutritional aspects to develop and yeasts biocompatibility.
